KLF6-SV1 drives breast cancer metastasis and is associated with poor survival

Abstract

Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node-negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal-like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.

Fig. 1

Increased KLF6-SV1SV1 expression in primary breast tumors correlates with poor prognosis. Five-year MFS as a function of KLF6-SV1 in 671 LNN primary breast cancer patients after trichotomizing KLF6-SV1 mRNA levels in three equal parts (low, intermediate, and high). Patients at risk are indicated.

Fig. 2

High levels of KLF6-SV1 mRNA associated with primary tumor characteristics. (A) KLF6-SV1 mRNA levels were correlated with stromal content (n = 453 LNN primary breast tumors with 30 to 70% invasive tumor cells versus n = 218 with more than 70% invasive tumor cells). (B) ESR1 (n = 492 ESR1-positive LNN primary breast tumors versus n = 179 ESR1-negative LNN primary breast tumors). (C) GGI (n = 218 GGI low LNN primary breast tumors versus n = 220 GGI intermediate LNN primary tumors versus n = 221 GGI high LNN primary breast tumors). mRNA levels were measured by qRT-PCR. The strengths of the associations between continuous variables were tested with a two-tailed Mann-Whitney U test.

Fig. 3

Overexpression of KLF6-SV1 promotes breast cancer development and progression in breast cell lines. (A) Colony assay of KLF6-SV1–expressing mammary epithelial cells stained at day 8; graphs represent the average number of colonies in pBABE control versus KLF6-SV1–expressing cells for MCF12A (P = 0.0027) and MCF10A (P = 0.0066). (B) Quantification of apoptosis in KLF6-SV1–expressing acini detected by cleaved caspase 3 staining by day 10 using indirect immunofluorescence (images taken at ×400 magnification). Acini containing two or more active caspase 3–positive cells were scored as positive, and 100 acini were counted per experiment (P = 0.0026). (C) Quantification of invasion and migration in Transwell chambers toward chemoattractant serum media over 24 hours in pBABE control versus KLF6-SV1–expressing cells in MCF12A migration (P = 0.034) and invasion (P = 0.002), MCF10A migration (P = 0.045) and invasion (P = 0.037), and BT474 migration (P = 0.011) and invasion (P = 0.0017). (D) Wound-healing assay over a period of 18 hours; graph represents percent wound closure over 12 hours for MCF12A (P = 0.006) and MCF10A (P = 0.007); images were taken at ×200 magnification. (E) Expression of epithelial marker E-cadherin and mesenchymal marker N-cadherin and fibronectin-expressing cells detected by Western blot. (F) Immunocytochemistry for N-cadherin and E-cadherin in pBABE versus p-SV1 cells taken at ×400 magnification. (G) KLF6-SV1–expressing MCF10A multiacinar structures during morphogenesis at day 20, taken at ×200 and ×400 magnifications. Representative images are demonstrated here and were repeated in triplicate; 1:100 of each antibody was used for staining. Data are represented as means ± SD of triplicate experiments.

Fig. 4

Silencing KLF6-SV1 in metastatic cell line reverts cells back to an epithelial-like expression pattern. (A) Western blot analysis of wtKLF6, KLF6-SV1, N-cadherin, and E-cadherin expression after siRNA-mediated knockdown of KLF6-SV1 in MDA-MB-231-luc-D3H2LN cell line. (B) Gene expression analysis of KLF6-SV1 (P < 0.001) and E-cadherin (P = 0.002) analyzed by qRT-PCR. (C) Invasion (P < 0.001) and migration (P = 0.003) toward serum media over 24 hours. (D) Wound-healing assay from cells grown on a monolayer over 24 hours; graph represents percent wound closure over a period of 24 hours; images were taken at ×200 magnification (P = 0.007). Data are represented as means ± SD of triplicate experiments.

Fig. 5

KLF6-SV1 induces metastasis in a subcutaneous mouse model. (A) Tumor volume of BT474 pBABE-expressing (n = 8) compared to p-SV1–expressing (n = 9) tumors. (B) Representative H&E staining of pBABE and p-SV1 tumors, with arrow pointing to local invasion in the p-SV1 tumor. (C) Representative images and H&E-stained sections of cardiac myocytes of the heart, kidney, lung, liver, spleen, and intestinal tract from a BT474 KLF6-SV1 mouse. Arrows point to metastatic lesions. (D) Sites of metastasis in BT474 pBABE- versus p-SV1–injected mice in the KLF6-SV1 mice (P < 0.001) analyzed by χ² analysis. All data are represented as means ± SD.

Fig. 6

Overexpression of KLF6-SV1 in an orthotopic model of breast cancer progression enhances metastasis. (A) Female nu/nu mice injected orthotopically with MDA-MB-231-luc-D3H2LN pBABE (n = 9) or p-SV1 (n = 8) cells into mammary fat pad; representative luminescent images from each group at day 21 and bioluminescence graph of local tumor growth for 27 days. (B) Tumor burden of pBABE (n = 9) and p-SV1 (n = 8) mammary tumors from mice sacrificed at day 27. (C) Representative ex vivo imaging of lung and liver from each group. (D) Representative H&E-stained lung section, with arrow pointing at a metastatic lung lesion. (E) Percentage of mice with metastasis observed using ex vivo analysis, lung metastasis (P = 0.006), and total metastasis (P = 0.072). Data are represented as means ± SD. P value was obtained by χ² analysis.

Fig. 7

Increased KLF6-SV1 up-regulates TWIST1 overexpression. (A) Expression of TWIST1 analyzed by Western blot in MCF12A, MCF10A, and BT474 cell lines. (B and C) Gene expression analysis of TWIST1 by qRT-PCR in (B) MCF12A (P = 0.0018), MCF10A (P = 0.0011), and BT474 (P = 0.0048) cell lines and (C) BT474 tumors (P = 0.002). (D) Immunohistochemical analysis of TWIST1 in pBABE and p-SV1 BT474 tumors demonstrating increased TWIST1 staining at ×400 magnification. Data are represented as means ± SD. Whiskers represent minimum and maximum values. Experiments were repeated in triplicate. Tumors were analyzed with Mann-Whitney statistical analysis.